Aggregated Myocilin Induces Russell Bodies and Causes Apoptosis
2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060806
ISSN1525-2191
AutoresGary Hin‐Fai Yam, Katarína Gaplovská-Kyselá, Christian Zuber, Jürgen Roth,
Tópico(s)Mitochondrial Function and Pathology
ResumoPrimary open-angle glaucoma with elevated intraocular pressure is a leading cause of blindness worldwide. Mutations of myocilin are known to play a critical role in the manifestation of the disease. Misfolded mutant myocilin forms secretion-incompetent intracellular aggregates. The block of myocilin secretion was proposed to alter the extracellular matrix environment of the trabecular meshwork, with subsequent impediment of aqueous humor outflow leading to elevated intraocular pressure. However, the molecular pathogenesis of myocilin-caused glaucoma is poorly defined. In this study, we show that heteromeric complexes composed of wild-type and mutant myocilin were retained in the rough endoplasmic reticulum, aggregating to form inclusion bodies typical of Russell bodies. The presence of myocilin aggregates induced the unfolded protein response proteins BiP and phosphorylated endoplasmic reticulum-localized eukaryotic initiation factor-2α kinase (PERK) with the subsequent activation of caspases 12 and 3 and expression of C/EBP homologous protein (CHOP)/GADD153, leading to apoptosis. Our findings identify endoplasmic reticulum stress-induced apoptosis as a pathway to explain the reduction of trabecular meshwork cells in patients with myocilin-caused glaucoma. As a consequence, the phagocytotic capacity of the remaining trabecular meshwork cell population would be insufficient for effective cleaning of aqueous humor, constituting a major pathogenetic factor for the development of increased intraocular pressure in primary open-angle glaucoma. Primary open-angle glaucoma with elevated intraocular pressure is a leading cause of blindness worldwide. Mutations of myocilin are known to play a critical role in the manifestation of the disease. Misfolded mutant myocilin forms secretion-incompetent intracellular aggregates. The block of myocilin secretion was proposed to alter the extracellular matrix environment of the trabecular meshwork, with subsequent impediment of aqueous humor outflow leading to elevated intraocular pressure. However, the molecular pathogenesis of myocilin-caused glaucoma is poorly defined. In this study, we show that heteromeric complexes composed of wild-type and mutant myocilin were retained in the rough endoplasmic reticulum, aggregating to form inclusion bodies typical of Russell bodies. The presence of myocilin aggregates induced the unfolded protein response proteins BiP and phosphorylated endoplasmic reticulum-localized eukaryotic initiation factor-2α kinase (PERK) with the subsequent activation of caspases 12 and 3 and expression of C/EBP homologous protein (CHOP)/GADD153, leading to apoptosis. Our findings identify endoplasmic reticulum stress-induced apoptosis as a pathway to explain the reduction of trabecular meshwork cells in patients with myocilin-caused glaucoma. As a consequence, the phagocytotic capacity of the remaining trabecular meshwork cell population would be insufficient for effective cleaning of aqueous humor, constituting a major pathogenetic factor for the development of increased intraocular pressure in primary open-angle glaucoma. Glaucoma is a heterogeneous group of optic neuropathies characterized by a progressive degeneration of the optic nerve resulting in an irreversible loss of vision.1Shields MB Ritch R Krupin T Classification of the Glaucoma.in: Ritch R Shields MB Krupin T Mosby, Philadelphia1996: 717-725Google Scholar After age-related macular degeneration, it is the second leading cause of severe vision loss or blindness.2Quigley HA Number of people with glaucoma worldwide.Br J Ophthalmol. 1996; 80: 389-393Crossref PubMed Scopus (1960) Google Scholar It is expected that ∼4% of the world population older than the age of 40 will develop glaucoma. Primary open-angle glaucoma (POAG) is the most common form of the disease.2Quigley HA Number of people with glaucoma worldwide.Br J Ophthalmol. 1996; 80: 389-393Crossref PubMed Scopus (1960) Google Scholar In most cases of POAG, the aqueous humor outflow from the anterior eye chamber is impeded, resulting in an elevated intraocular pressure (IOP) and causing ganglion cell death in the neural retina.3Tamm ER Myocilin and glaucoma: facts and ideas.Prog Retin Eye Res. 2002; 21: 395-428Crossref PubMed Scopus (175) Google Scholar, 4Tan JC Peters DM Kaufman PL Recent developments in understanding the pathophysiology of elevated intraocular pressure.Curr Opin Ophthalmol. 2006; 17: 168-174PubMed Google Scholar Hence, impaired outflow drainage along the trabecular meshwork (TM) and Schlemm's canal seems to be central in the pathogenesis of POAG.3Tamm ER Myocilin and glaucoma: facts and ideas.Prog Retin Eye Res. 2002; 21: 395-428Crossref PubMed Scopus (175) Google Scholar, 4Tan JC Peters DM Kaufman PL Recent developments in understanding the pathophysiology of elevated intraocular pressure.Curr Opin Ophthalmol. 2006; 17: 168-174PubMed Google Scholar The flow of aqueous humor is important in providing nutritive support for the avascular anterior eye tissues, such as cornea and lens. The aqueous humor is constitutively produced by the ciliary body, enters the anterior eye chamber, and exits it through the TM, a reticulated network of cell-lined extracellular matrix located at the junction of cornea and iris, enters the Schlemm's canal, and finally the venous circulation.5Grierson I Hogg P The proliferative and migratory activities of trabecular meshwork cells.Prog Retin Eye Res. 1995; 15: 33-67Crossref Scopus (38) Google Scholar The cells of the TM seem to play a regulatory role in humor outflow. TM cells synthesize and release glycosaminoglycans, glycoproteins, and fibrillar materials6Acott TS Westcott M Passo MS van Buskirk EM Trabecular meshwork glycosaminoglycans in human and cynomolgus monkey eye.Invest Ophthalmol Vis Sci. 1985; 26: 1320-1329PubMed Google Scholar and are active as phagocytes.7Buller C Johnson DH Tschumper RC Human trabecular meshwork phagocytosis. Observations in an organ culture system.Invest Ophthalmol Vis Sci. 1990; 31: 2156-2163PubMed Google Scholar By phagocytosing particulate matter of various origins, TM cells help to maintain the structural and functional integrity of the TM drainage pathway. Different organotypic and cell culture models have been used to investigate the role of TM cells in the outflow drainage. Disruption of the actin cytoskeleton by inhibition of Rho kinase in organ culture of anterior eye segment reduces the focal adhesion of TM cells and increases aqueous humor outflow.8Rao PV Deng P Maddala R Epstein DL Li CY Shimokawa H Expression of dominant negative Rho-binding domain of Rho-kinase in organ cultured human eye anterior segments increases aqueous humor outflow.Mol Vis. 2005; 11: 288-297PubMed Google Scholar Expression of recombinant Hep II domain of fibronectin has also found to increase the outflow.9Santas AJ Bahler C Peterson JA Filla MS Kaufman PL Tamm ER Johnson DH Peters DM Effect of heparin II domain of fibronectin on aqueous outflow in cultured anterior segments of human eyes.Invest Ophthalmol Vis Sci. 2003; 44: 4796-4804Crossref PubMed Scopus (51) Google Scholar In contrast, dexamethasone-induced actin cross-linking reduces aqueous humor outflow in a perfusion model of isolated anterior segment.10Clark AF Brotchie D Read AT Hellberg P English-Wright S Pang IH Ethier CR Grierson I Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissues.Cell Motil Cytoskeleton. 2005; 60: 83-95Crossref PubMed Scopus (160) Google Scholar These data point to a role of cell-extracellular matrix interactions in the outflow drainage. On the other hand, mutations of the myocilin (MYOC) gene have been found in most families with juvenile-onset POAG and in ∼4% of patients with adult-onset POAG. MYOC was the first identified glaucoma-causing gene,11Stone EM Fingert JH Alward LM Nguyen TD Polansky JR Sunden SL Nishimura D Clark AF Nystuen A Nichols BE Mackey DA Ritch R Kalenak JW Identification of a gene that causes primary open angle glaucoma.Science. 1997; 275: 668-670Crossref PubMed Scopus (1242) Google Scholar and the majority of patients with MYOC mutations suffer from high IOP.12Alward WL Fingert JH Coote MA Johnson AT Lerner SF Junqua D Durcan FJ McCartney PJ Mackey DA Sheffield VC Stone EM Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A).N Engl J Med. 1998; 338: 1022-1027Crossref PubMed Scopus (400) Google Scholar MYOC is synthesized and secreted by TM cells13Swiderski RE Ross JL Fingert JH Clark AF Alward WL Stone EM Sheffield VC Localization of MYOC transcripts in human eye and optic nerve by in situ hybridization.Invest Ophthalmol Vis Sci. 2000; 41: 3420-3428PubMed Google Scholar, 14Huang W Jaroszewski J Ortego J Escribano J Coca-Prados M Expression of the TIGR gene in the iris, ciliary body, and trabecular meshwork of the human eye.Ophthalmic Genet. 2000; 21: 155-169Crossref PubMed Google Scholar, 15Karali A Russell P Stefani F Tamm E Localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein in the human eye.Invest Ophthalmol Vis Sci. 2000; 41: 729-740PubMed Google Scholar, 16Tamm ER Polansky JR The TIGR/MYOC gene and glaucoma: opportunities for new understandings.J Glaucoma. 2001; 10: S9-S12Crossref PubMed Scopus (11) Google Scholar and has been found to be associated with different microfibrillar structures and with sheath-derived plaque material in the TM.17Ueda J Wentz-Hunter K Yue BY Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes.Invest Ophthalmol Vis Sci. 2002; 43: 1068-1076PubMed Google Scholar, 18Fautsch MP Vrabel AM Johnson DH The identification of myocilin-associated proteins in the human trabecular meshwork.Exp Eye Res. 2006; 82: 1046-1052Crossref PubMed Scopus (34) Google Scholar A recent functional analysis has shown the importance of the olfactomedin-homology domain of MYOC for its secretion and that of the amino-terminal coiled-coil regions for the interactions of MYOC with extracellular matrix components.19Gobeil S Letartre L Raymond V Functional analysis of the glaucoma-causing TIGR/myocilin protein: integrity of amino-terminal coiled-coil regions and olfactomedin homology domain is essential for extracellular adhesion and secretion.Exp Eye Res. 2006; 82: 1017-1029Crossref PubMed Scopus (77) Google Scholar The molecular pathogenesis of MYOC-caused POAG is still poorly defined.20Whitmore AV Libby RT John SW Glaucoma: thinking in new ways—a role for autonomous axonal self-destruction and other compartmentalized processes?.Prog Retin Eye Res. 2005; 24: 639-662Crossref PubMed Scopus (195) Google Scholar Absence21Kim BS Savinova OV Reedy MV Martin J Lun Y Gan L Smith RS Tomarev SI John SW Johnson RL Targeted disruption of the myocilin gene (Myoc) suggests that human glaucoma-causing mutations are gain of function.Mol Cell Biol. 2001; 21: 7707-7713Crossref PubMed Scopus (218) Google Scholar or overexpression22Gould DB Miceli-Libby L Savinova OV Torrado M Tomarev SI Smith RS John SWM Genetically increasing Myoc expression supports a necessary pathologic role of abnormal proteins in glaucoma.Mol Cell Biol. 2004; 24: 9019-9025Crossref PubMed Scopus (105) Google Scholar of wild-type (WT) MYOC in transgenic mice has been shown not to be critical for the development of POAG. However, many glaucoma-causing mutant MYOCs have been found to be misfolded and to form detergent-resistant, secretion-incompetent aggregates.19Gobeil S Letartre L Raymond V Functional analysis of the glaucoma-causing TIGR/myocilin protein: integrity of amino-terminal coiled-coil regions and olfactomedin homology domain is essential for extracellular adhesion and secretion.Exp Eye Res. 2006; 82: 1017-1029Crossref PubMed Scopus (77) Google Scholar, 23Zhou Z Vollrath D A cellular assay distinguishes normal and mutant TIGR/myocilin protein.Hum Mol Genet. 1999; 8: 2221-2228Crossref PubMed Scopus (112) Google Scholar, 24Jacobson N Andrews M Shepard AR Nishimura D Searby C Fingert JH Hageman G Mullins R Davidson BL Kwon YH Alward WL Stone EM Clark AF Sheffield VC Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.Hum Mol Genet. 2001; 10: 117-125Crossref PubMed Scopus (245) Google Scholar, 25Joe MK Sohn S Hur W Moon Y Choi YR Kee C Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells.Biochem Biophys Res Commun. 2003; 312: 592-600Crossref PubMed Scopus (179) Google Scholar, 26Liu Y Vollrath D Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma.Hum Mol Genet. 2004; 13: 1193-1204Crossref PubMed Scopus (179) Google Scholar, 27Vollrath D Liu YH Temperature sensitive secretion of mutant myocilins.Exp Eye Res. 2006; 82: 1030-1036Crossref PubMed Scopus (43) Google Scholar The secretion block of POAG-causing mutant MYOCs could be overcome by lowering the cell culture temperature from 37 to 30°C, a condition known to improve protein folding.17Ueda J Wentz-Hunter K Yue BY Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes.Invest Ophthalmol Vis Sci. 2002; 43: 1068-1076PubMed Google Scholar, 19Gobeil S Letartre L Raymond V Functional analysis of the glaucoma-causing TIGR/myocilin protein: integrity of amino-terminal coiled-coil regions and olfactomedin homology domain is essential for extracellular adhesion and secretion.Exp Eye Res. 2006; 82: 1017-1029Crossref PubMed Scopus (77) Google Scholar Increasing evidence suggests that mutant MYOC acts by a pathological gain-of-function mechanism.21Kim BS Savinova OV Reedy MV Martin J Lun Y Gan L Smith RS Tomarev SI John SW Johnson RL Targeted disruption of the myocilin gene (Myoc) suggests that human glaucoma-causing mutations are gain of function.Mol Cell Biol. 2001; 21: 7707-7713Crossref PubMed Scopus (218) Google Scholar, 28Morissette J Clepet C Moisan S Dubois S Winstall E Vermeeren D Nguyen TD Polansky JR Cote G Anctil JL Amyot M Plante M Falardeau P Raymond V Homozygotes carrying an autosomal dominant TIGR mutation do not manifest glaucoma.Nat Genet. 1998; 19: 319-321Crossref PubMed Scopus (120) Google Scholar, 29Lam DS Leung YF Chua JK Baum L Fan DS Choy KW Pang CP Truncations in the TIGR gene in individuals with and without primary open-angle glaucoma.Invest Ophthalmol Vis Sci. 2000; 41: 1386-1391PubMed Google Scholar In agreement with this, several missense and truncated mutant MYOCs have been shown to interact with WT MYOCs to form heteromeric protein aggregates resulting in a block of secretion of WT MYOCs as well.25Joe MK Sohn S Hur W Moon Y Choi YR Kee C Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells.Biochem Biophys Res Commun. 2003; 312: 592-600Crossref PubMed Scopus (179) Google Scholar, 30Sohn S Hur W Joe MK Kim JH Lee ZW Ha HS Kee C Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities.Invest Ophthalmol Vis Sci. 2002; 43: 3680-3685PubMed Google Scholar, 31Gobeil S Rodrigue MA Moisan S Nguyen TD Polansky JR Morissette J Raymond V Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants.Invest Ophthalmol Vis Sci. 2004; 45: 3560-3567Crossref PubMed Scopus (88) Google Scholar MYOC-secreting TM cells are phagocytotically active and keep the outflow drainage along the TM clean of deposits of particulate matter of various origin.7Buller C Johnson DH Tschumper RC Human trabecular meshwork phagocytosis. Observations in an organ culture system.Invest Ophthalmol Vis Sci. 1990; 31: 2156-2163PubMed Google Scholar, 32Shirato S Murphy CG Bloom E Franse-Carman L Maglio MT Polansky JR Alvarado JA Kinetics of phagocytosis in trabecular meshwork cells. Flow cytometry and morphometry.Invest Ophthalmol Vis Sci. 1989; 30: 2499-2511PubMed Google Scholar, 33Schlotzer-Schrehardt U Naumann GO Trabecular meshwork in pseudoexfoliation syndrome with and without open-angle glaucoma. A morphometric, ultrastructural study.Invest Ophthalmol Vis Sci. 1995; 36: 1750-1764PubMed Google Scholar, 34Matsumoto Y Johnson DH Dexamethasone decreases phagocytosis by human trabecular meshwork cells in situ.Invest Ophthalmol Vis Sci. 1997; 38: 1902-1907PubMed Google Scholar We reasoned that a disturbance of the phagocytotic capacity of the TM cell population could be a pathogenetic factor in the development of MYOC-caused POAG. This is supported by electron microscopic findings of thickened trabeculae and the accumulation of sheath-derived plaques and of melanin.3Tamm ER Myocilin and glaucoma: facts and ideas.Prog Retin Eye Res. 2002; 21: 395-428Crossref PubMed Scopus (175) Google Scholar, 17Ueda J Wentz-Hunter K Yue BY Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes.Invest Ophthalmol Vis Sci. 2002; 43: 1068-1076PubMed Google Scholar, 35Lütjen-Drecoll E Shimizu T Rohrbach M Rohen JW Quantitative analysis of ‘plaque material’ in the inner- and outer wall of Schlemm's canal in normal- and glaucomatous eyes.Exp Eye Res. 1986; 42: 443-455Crossref PubMed Scopus (218) Google Scholar, 36Rohen JW Lütjen-Drecoll E Flugel C Meyer M Grierson I Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG).Exp Eye Res. 1993; 56: 683-692Crossref PubMed Scopus (156) Google Scholar, 37Gottanka J Flugel-Koch C Martus P Johnson DH Lütjen-Drecoll E Correlation of pseudoexfoliative material and optic nerve damage in pseudoexfoliation syndrome.Invest Ophthalmol Vis Sci. 1997; 38: 2435-2446PubMed Google Scholar, 38Lütjen-Drecoll E Functional morphology of the trabecular meshwork in primate eyes.Prog Retin Eye Res. 1999; 18: 91-119Crossref PubMed Scopus (217) Google Scholar, 39Cracknell KP Grierson I Hogg P Majekodunmi AA Watson P Marmion V Melanin in the trabecular meshwork is associated with age, POAG but not Latanoprost treatment. A masked morphometric study.Exp Eye Res. 2006; 82: 986-993Crossref PubMed Scopus (17) Google Scholar Furthermore, a reduction of the number of TM cells has been observed in glaucoma patients.36Rohen JW Lütjen-Drecoll E Flugel C Meyer M Grierson I Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG).Exp Eye Res. 1993; 56: 683-692Crossref PubMed Scopus (156) Google Scholar, 40Alvarado J Murphy C Juster R Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals.Ophthalmology. 1984; 91: 564-579Abstract Full Text PDF PubMed Scopus (465) Google Scholar Although mutant MYOC singly expressed in different cell types has been shown to be cytotoxic,25Joe MK Sohn S Hur W Moon Y Choi YR Kee C Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells.Biochem Biophys Res Commun. 2003; 312: 592-600Crossref PubMed Scopus (179) Google Scholar, 26Liu Y Vollrath D Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma.Hum Mol Genet. 2004; 13: 1193-1204Crossref PubMed Scopus (179) Google Scholar, 30Sohn S Hur W Joe MK Kim JH Lee ZW Ha HS Kee C Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities.Invest Ophthalmol Vis Sci. 2002; 43: 3680-3685PubMed Google Scholar its mechanism of action remains elusive. The possible cytotoxicity of heteromeric mutant/WT MYOC complexes whose formation is the basis for the pathological gain-of-function mechanism has not been addressed yet. In the present study, we provide evidence for a link between the formation of heteromeric mutant/WT MYOC aggregates, endoplasmic reticulum (ER) stress, and apoptosis. In a cell culture system, we demonstrate Russell body formation by ER-retained heteromeric mutant/WT MYOC aggregates, which induce ER stress and lead to apoptosis. This supports the proposal that the phagocytotic capacity of the reduced TM cell population is insufficient for effective cleaning of the TM. This may represent an important pathogenetic factor for the development of increased IOP in patients with POAG. Chinese hamster ovary (CHO)-K1 and human embryonic kidney (HEK) 293 cells were purchased from American Type Culture Collection (Manassas, VA) and immortalized human TM (HTM) cells41Polansky JR Weinreb RN Baxter JD Alvarado J Human trabecular cells. I. Establishment in tissue culture and growth characteristics.Invest Ophthalmol Vis Sci. 1979; 18: 1043-1049PubMed Google Scholar were kindly provided by T.D. Nguyen (San Francisco, CA). MYOC mRNA or MYOC protein was undetectable in the human TM cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively (data not shown). Culture media, Lipofectamine 2000, protein A Dynabeads, fetal bovine serum, normal goat serum, and Geneticin 418 were from Invitrogen (Basel, Switzerland), Fugene 6 and protease inhibitor cocktail from Roche Diagnostics (Rotkreuz, Switzerland), expression vectors pEGFP-N3 from Clontech (Basel, Switzerland), QuikChange II site-directed mutagenesis kit from Stratagene (La Jolla, CA), and p3xFLAG-myc-CMV-25 from Sigma (Buchs, Switzerland). Oligonucleotides were synthesized by MicroSynth (Balgach, Switzerland). MG132 was from Calbiochem (La Jolla, CA), enhanced chemiluminescence Western blotting detection kit from Amersham Biosciences (Buckinghamshire, UK), [35S]cysteine and [35S]methionine from Anawa (Wangen, Switzerland), and EN3hance from Perkin-Elmer (Boston, MA). Mouse monoclonal antibodies against the ER chaperone BiP (BD Biosciences, San Jose, CA), the pre-Golgi intermediate marker ERGIC-53 (a gift from H.P. Hauri, Biozentrum, University of Basel, Basel, Switzerland), GAPDH (Ambion, Austin, TX), and β-actin (Sigma) and rabbit polyclonal antibodies against green fluorescent protein (GFP; Molecular Probes, Eugene, OR), FLAG (Sigma), phosphorylated ER-localized eukaryotic initiation factor (eIF)-2α kinase (PERK; Santa Cruz Biotechnology Inc., Santa Cruz, CA), C/EBP homologous protein (CHOP)/GADD153 (Alexis, Lausen, Switzerland), caspase 3 (Upstate, Lake Placid, NY), and caspase 12 (ProSci, Poway, CA) were used. Alexa 488-conjugated goat anti-rabbit Ig antibody (Molecular Probes), red X-conjugated Fab fragments of goat anti-mouse Ig or anti-rabbit Ig (Jackson ImmunoResearch, West Grove, PA), and horseradish peroxidase-conjugated donkey anti-rabbit Ig or sheep anti-mouse Ig antibodies (Amersham Biosciences) were used. Bovine serum albumin, Triton X-100 and diaminobenzidine tetrahydrochloride (DAB) were from Sigma, glutaraldehyde and osmium tetroxide from EMD (Gibbstown, NJ), formaldehyde from Merck (Basel, Switzerland), and Epon-Araldite from Fluka (Buchs, Switzerland). All other chemicals were of analytical grade and from Sigma. Human full-length MYOC cDNA was obtained from human skeletal muscle and cloned into the expression vector pEGFP-N3 and p3xFLAG-myc-CMV-25 to generate the constructs pEGFP-MYOCWT and pFLAG-MYOCWT, respectively. Mutations in MYOC cDNA were introduced by a PCR-based method using QuikChange II site-directed mutagenesis kit and specific primers. The sense primer sequences with the altered bases underlined for C245Y MYOC were 5′-GAGTGGAGAGGGAGACACCGGATATGGAGAACTAGTTTGGGTAGG-3′; for G364V MYOC: GAAGGAAATCCCTGGAGCTGTCTACCACGGACAGTTCCCG-3′; for P370L MYOC: 5′-CTGGCTACCACGGACAGTTCCTGTATTCTTGGGGTGGCTACACG-3′; for R422C MYOC: 5′-CAAACCTGGGAGACAAACATCT-GTAAGCAGTCAGTCGCCAATGCC-3′; for R422H MYOC: 5′-CAAACCTGGGAGACAAACATCCATAAGCAGTCAGTCGCCAATGCC-3′; and for Y437H MYOC: 5′-CATCATCTGTGGCACCTTGCACACCGTCAGCAGCTACACC-3′. The correctness of all constructs was verified by direct sequencing. CHO-K1 and HEK293 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum, 110 μg/ml sodium pyruvate, and antibiotics. HTM cells were grown in Dulbecco's modified Eagle's medium (1 mg/ml d-glucose) supplemented with fetal bovine serum, sodium pyruvate, and antibiotics. Cells at ∼50% confluence were transfected with plasmids containing WT or different mutant MYOC cDNAs by Fugene 6 or Lipofectamine 2000 according to the manufacturer's protocol with a ratio of 3 μl of transfection reagent per μg of DNA. For stable MYOC expression, the cells were selected with 100 μg/ml Geneticin 418 in culture medium for 10 days, and cells with suitable MYOC expression level were clonally expanded. For double MYOC expression, cells stably expressing MYOC-GFP fusion protein were transiently transfected with pFLAG-MYOCWT and analyzed within 5 days. Cells were incubated with media containing 25 μCi/ml [35S]cysteine and [35S]methionine for 24 hours. Culture media were collected and centrifuged to remove cell debris. The supernatant was immunoprecipitated with polyclonal antibodies against FLAG bound to protein A Dynabeads. Immunoprecipitated proteins were denatured in sodium dodecyl sulfate (SDS) sample buffer containing 20% β-mercaptoethanol and resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE). The gel was treated with EN3hance, and radioactivity was detected by a Bio-Imaging analyzer (BAS-1800II; Fujifilm, Wayne, NJ). Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in buffer containing 100 mmol/L Tris-HCl (pH 7.4), 3 mmol/L EGTA, 5 mmol/L MgCl2, 0.5% Triton X-100, protease inhibitor cocktail, and 1 mmol/L phenylmethyl sulfonyl fluoride for 2 minutes on ice.23Zhou Z Vollrath D A cellular assay distinguishes normal and mutant TIGR/myocilin protein.Hum Mol Genet. 1999; 8: 2221-2228Crossref PubMed Scopus (112) Google Scholar After centrifugation, the pellet containing Triton X-100-insoluble proteins was washed with ice-cold PBS twice, sonicated, and denatured in SDS sample buffer containing 9 mol/L urea. Triton X-100-insoluble proteins from samples equivalent to 2 × 105 cells were analyzed by 10% SDS-PAGE and Western blotting using antibodies against GFP, FLAG, or β-actin, respectively, and appropriate horseradish peroxidase-conjugated secondary antibodies. The signals were detected by enhanced chemiluminescence. MYOC-GFP-expressing cells grown to ∼70% confluence were incubated in cysteine- and methionine-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum for 5 minutes at 37°C. For metabolic labeling, the cells were incubated in fresh medium containing 100 μCi/ml [35S]cysteine and [35S]methionine for 8 minutes. After two rinses with PBS, the cells were cultured in fresh medium supplemented with 1 mmol/L nonradioactive cysteine and methionine in the presence or not of 10 μmol/L MG132 for periods ranging from 1 to 8 hours. The cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, protease inhibitor cocktail, and 1 mmol/L phenylmethyl sulfonyl fluoride for 30 minutes on ice. After centrifugation, the supernatant was immunoprecipitated for GFP and analyzed. Cells expressing WT or co-expressing mutant MYOC-GFP and FLAG-WT MYOC were lysed in RIPA buffer. To study PERK expression, phosphatase inhibitors (1 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, and 10 mmol/L sodium diphosphate) were added to the RIPA buffer. After centrifugation, the supernatant was denatured in SDS sample buffer containing 50 mmol/L dithiothreitol and samples (20 μg) were resolved by 8 or 12% SDS-PAGE followed by incubation with antibodies against BiP, PERK, caspase 12, CHOP/GADD153, caspase 3, FLAG, GFP, or GAPDH and appropriate horseradish peroxidase-conjugated secondary antibodies. Signals were detected by enhanced chemiluminescence. For conventional transmission electron microscopy, cells were fixed in situ with 3% glutaraldehyde followed by 1% osmium tetroxide and embedded in Epon-Araldite according to standard protocol. Ultrathin sections were prepared using an Ultracut S ultramicrotome (Leica, Wetzlar, Germany), contrasted with 1% uranyl acetate and lead citrate and examined with an EM912 AB transmission electron microscope (Zeiss, Oberkochen, Germany). Subcellular distribution of MYOC in mutant/WT MYOC-expressing HEK293 cells was analyzed by pre-embedding immunoperoxidase electron microscopy.42Brown WJ Farquhar MG Immunoperoxidase methods for the localization of antigens in cultured cells and tissue sections by electron microscopy.Methods Cell Biol. 1989; 31: 553-569Crossref PubMed Scopus (84) Google Scholar In brief, cells grown on glass coverslips were fixed with freshly prepared 2% formaldehyde in phosphate buffer (pH 7.4, 0.15 mol/L NaCl) for 10 minutes at 37°C, permeabilized with 0.05% saponin in PBS containing 1.5% normal goat serum and 0.1% bovine serum albumin for 10 minutes, incubated with polyclonal antibody to FLAG followed by horseradish peroxidase-conjugated secondary antibody, both diluted in PBS containing 1% bovine serum albumin and 0.05% saponin. Afterward, cells were washed in 0.1 mol/L cacodylate buffer (pH 7.4), postfixed in 2% buffered glutaraldehyde for 1 hour. Peroxidase activity was revealed by the DAB reaction. DAB (0.2%) was dissolved in 50 mmol/L Tris-HCl, pH 7.4, containing 7.5% sucrose to which H2O2 was added (final concentration, 0.002%). Coverslips were incubated with the DAB solution for 17 minutes. After washing, samples were fixed with 1% reduced osmium tetroxide in cacodylate buffer, dehydrated in a series of graded ethanol, and embedded in situ in Epon-Araldite. Ultrathin sections were prepared parallel to the plane of the cell monolayer, contrasted, and examined by transmission electron microscopy. HEK293 cells co-expressing WT or mutant MYOC-GFP and FLAG-WT MYOC were grown o
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